(Related to Figs. 4 and 6). (A) Representative image from a culture with 1:1 ratio of mOC control (CTL, transduced with mCherry-lifeact) and moesin KO (transduced with GFP-lifeact) seeded on glass coverslips on day 4 of differentiation. Arrowheads show green podosome belts. Scale bar, 20 µm. (B) Effect of moesin KO on sealing zone formation in mOC. mOC control (CTL) versus moesin KO (MKO) were cultured for 5 days on glass coverslips, detached and then seeded for additional 2 days on bone slices. Quantification of the area occupied by sealing zones (left) and circularity of sealing zones (right). Each circle represents a single donor, n = 4, SDs are shown. (C) Effect of moesin depletion on sealing zone formation in hOC. 6 day–differentiated hOC on glass coverslips treated on day 0 with siCTL or siMoesin were detached and seeded for additional 24 h on bone slices. Quantification of the number of nuclei per cells forming sealing zones. Each circle represents an independent experiment, n = 4 donors. n.s., not significant. (A–C related to Fig. 4). (D) Effect of NSC23 and ML-141 on ERM activation. 6-day hOCs were treated or not (CTL) with NSC23 and ML 141, drugs targeting Rac1/2 and Cdc42 respectively. Representative western blot analysis (left) and quantification of P-ERM signal normalized to actin (right). Each circle represents a single donor (n = 5, SDs are shown). (E) Effect of Y27632 (ROCK kinase inhibitor) treatment on P-ERM signal. Representative western blot analysis (left) and quantification of P-ERM signal normalized to actin (right). Each circle represents a single donor, n = 6, SDs are shown. Predicted molecular weight are indicated on western blots. (D and E related to Fig. 6) Statistical analyses: (D and E) Friedman and Dunn’s multiple comparison tests. n.s. not significant. Source data are available for this figure: SourceData FS5.